Androgen receptor is a primary transcription factor involved in the proliferation of prostate cancer cells. Thus, hormone therapy using antiandrogens, such as bicalutamide, is a first-line treatment for the disease. Although hormone therapy initially reduces the tumor burden, many patients eventually relapse, developing tumors with acquired endocrine resistance. Elucidation of the molecular mechanisms underlying endocrine resistance is therefore a fundamental issue for the understanding and development of alternative therapeutics for advanced prostate cancer. In the present study, we performed short hairpin RNA (shRNA)-mediated functional screening to identify genes involved in bicalutamide-mediated effects on LNCaP prostate cancer cells. Among such candidate genes selected by screening using volcano plot analysis, ribosomal protein L31 (RPL31) was found to be essential for cell proliferation and cell-cycle progression in bicalutamide-resistant LNCaP (BicR) cells, based on small interfering RNA (siRNA)-mediated knockdown experiments. Of note, RPL31 mRNA is more abundantly expressed in BicR cells than in parental LNCaP cells, and clinical data from ONCOMINE and The Cancer Genome Altas showed that RPL31 is overexpressed in prostate carcinomas compared with benign prostate tissues. Intriguingly, protein levels of the tumor suppressor p53 and its targets, p21 and MDM2, were increased in LNCaP and BicR cells treated with RPL31 siRNA. We observed decreased degradation of p53 protein after RPL31 knockdown. Moreover, the suppression of growth and cell cycle upon RPL31 knockdown was partially recovered with p53 siRNA treatment. These results suggest that RPL31 is involved in bicalutamide-resistant growth of prostate cancer cells. The shRNA-mediated functional screen in this study provides new insight into the molecular mechanisms and therapeutic targets of advanced prostate cancer.